Current detection platforms are essentially designed to assay the protein expression level in the biological samples, but for enzymes, not just the expression level but the catalytic activity are also important to their functional role in the pathologic process. We have recently developed a quantum dot-bioluminescence resonance energy transfer (QD-BRET) nanosystem. When a mutant of Renilla luciferase (Luc8) and the QDs are in close proximity, the biochemical energy generated in the oxidation of its substrate, coelenterazine, by Luc8 can be transferred to the QDs through BRET, generating light emission from the QDs. Since this QD-BRET process is distance-dependent, it offers a unique detection platform to assay the enzyme activity. This QD-BRET nanosystem offers many advantages over existing platforms for bioimaging and biodetection. First, by eliminating the need for the external light input, QD-BRET avoids problems associated with fluorescence detection such as background fluorescence, direct acceptor excitation, and photobleaching. Second, it offers great sensitivity for detection. Third, existing detection technologies are subject to the interference from the hemoglobin absorbance, and require serum rather than whole blood analysis. QD-BRET is compatible with analysis of whole blood, thus circumventing the need for serum preparation. Fourth, the QD-BRET platform is amenable to multiplexing and will enable analysis of several targets in a single test. In addition, by avoiding external illumination sources, this technology is much easier to be miniaturized and to be developed into portable diagnostic devices. Fifth, in comparison to the traditional FRET protocols using QDs, QD-BRET uses QD as the emitter which utilizes their outstanding absorption cross-section and broad absorption spectrum. This R21 application seeks to further advance the QD-BRET technology by developing competitive QD- BRET (cQD-BRET) sensors for highly sensitive multiplex detection of enzyme targets in biological samples with three specific aims: 1) Establish the cQD-BRET sensor design for multiplex detection of proteases in a homogenous assay with serum, whole blood and tissue samples. 2) Multiplexed detection of protease activity on a microplate reader platform with immobilized antibodies. 3) Comparison of detection sensitivity with commercial ELISA assay. PUBLIC HEALTH RELEVANCE: The proposed research aims to develop a novel nanotechnology to detect critical enzymes that are implicated in tumor formation, migration and metastasis. This new nanotechnology will enable highly sensitive detection of these enzyme molecules in biological samples to help early detection of cancers and to advance our understanding of the differences of enzyme activity between tumor and normal tissues during cancer formation and metastasis.